Nestin-dependent mitochondria-ER contacts define stem Leydig cell differentiation to attenuate male reproductive ageing

Male reproductive system ageing is closely associated with deficiency in testosterone production due to loss of functional Leydig cells, which are differentiated from stem Leydig cells (SLCs). However, the relationship between SLC differentiation and ageing remains unknown. In addition, active lipid metabolism during SLC differentiation in the reproductive system requires transportation and processing of substrates among multiple organelles, e.g., mitochondria and endoplasmic reticulum (ER), highlighting the importance of interorganelle contact. Here, we show that SLC differentiation potential declines with disordered intracellular homeostasis during SLC senescence. Mechanistically, loss of the intermediate filament Nestin results in lower differentiation capacity by separating mitochondria-ER contacts (MERCs) during SLC senescence. Furthermore, pharmacological intervention by melatonin restores Nestin-dependent MERCs, reverses SLC differentiation capacity and alleviates male reproductive system ageing. These findings not only explain SLC senescence from a cytoskeleton-dependent MERCs regulation mechanism, but also suggest a promising therapy targeting SLC differentiation for age-related reproductive system diseases.

(c) Flow cytometry analysis of apoptosis of primary Nestin-GFP+ SLCs from different age groups in vitro, double-stained with Annexin V-FITC and propidium iodine (PI).
(d) Quantification of apoptosis ratio of total cells from (e). (n = 3 biological repeats for each group; All data are mean ± SD; One-way ANOVA).
(e) Flow cytometry analysis of mitochondrial membrane potential stained with TMRE of primary Nestin-GFP+ SLCs from different age groups in vitro. 20 μM FCCP was the positive control added to the 2m group.
(f) Quantification of mean fluorescence intensity of TMRE in (g). (n = 3 biological repeats for each group; All data are mean ± SD; One-way ANOVA).
(g-i) Western Blot analysis and quantification of autophagy related proteins in Control (Vehicle) and Bafilomycin A1 (Baf A1) treated primary Nestin-GFP+ SLCs from different age groups in vitro. (n = 3 biological repeats for each group; All data are mean ± SD; One-way ANOVA).
(j) Representative immunostaining pictures of mitophagy in Control (Vehicle) and Bafilomycin A1 (Baf A1) treated primary Nestin-GFP+ SLCs from different age groups in vitro. Mitochondria are stained with Mitotracker and autophagy is stained with LC3. Scale bar, 10 μm.
(k) Quantitative analysis of mitophagy by calculating Mander's coefficient in (I).
(Manders' coefficient of mitochondria is shown for each condition, n = 48, 53 and 48 cells for 2m, 12m and 24m SLCs, respectively; All data are mean ± SD; One-way ANOVA).
(l-n) Western Blot analysis and quantification of mitophagy related proteins in primary Nestin-GFP+ SLCs from different age groups in vitro. (n = 3 biological repeats for each group; All data are mean ± SD; Multiple t tests).
(o) Quantitative analysis of mitochondrial ATP production in primary Nestin-GFP+ SLCs from different age groups in vitro (n = 3 biological repeats for each group; All data are mean ± SD; One-way ANOVA).
(p) Quantitative analysis of glucose uptake primary Nestin-GFP+ SLCs from different age groups in vitro (n = 3 biological repeats for each group; All data are mean ± SD; One-way ANOVA).
(q) Quantitative analysis of lactate production of primary Nestin-GFP+ SLCs from different age groups in vitro (n = 3 biological repeats for each group; All data are mean ± SD; One-way ANOVA). Two-sided comparison; Error bars represent SDs. *p < 0.05, **p < 0.01, ***p < 0.001; Uncropped western blots and source data are provided as a Source Data file. (a) qPCR analysis of relative mRNA expression of MAMs resident proteins in primary Nestin-GFP+ SLCs from different age groups in vitro. (n = 3 biological repeats for each group; All data are mean ± SD; Multiple t tests).
(b-c) Western Blot analysis and quantification of MAMs resident proteins expression in primary Nestin-GFP+ SLCs from different age groups in vitro. (n = 3 biological repeats for each group; All data are mean ± SD; Multiple t tests).
(d-g) qPCR analysis of relative mRNA expression of IP3R2 (d), Western Blot analysis and quantification of IP3R2 protein expression (e) and qPCR analysis of relative mRNA expression of anti-oxidative (f) and ER stress (g) related genes of IP3R2-knockdown primary SLCs from 2 months old mice. (n = 3 biological repeats for each group; All data are mean ± SD; Multiple t tests).
(h-k) qPCR analysis of relative mRNA expression of MFN2 (h), Western Blot analysis and quantification of MFN2 protein expression (i) and qPCR analysis of relative mRNA expression of anti-oxidative (j) and ER stress (k) related genes of MFN2-knockdown primary SLCs from 2 months old mice. (n = 3 biological repeats for each group; All data are mean ± SD; h-i, unpaired t test; j-k, multiple t tests).
(l-o) qPCR analysis of relative mRNA expression of PACS2 (l), Western Blot analysis and quantification of PACS2 protein expression (m) and qPCR analysis of relative mRNA expression of anti-oxidative (n) and ER stress (o) related genes of PACS2-knockdown primary SLCs from 2 months old mice. (n = 3 biological repeats for each group; All data are mean ± SD; l-m, unpaired t test; n-o, multiple t tests).
(p-s) qPCR analysis of relative mRNA expression of GRP75 (p), Western Blot analysis and quantification of GRP75 protein expression (q) and qPCR analysis of relative mRNA expression of anti-oxidative (r) and ER stress (s) related genes of GRP75-knockdown primary SLCs from 2 months old mice. (n = 3 biological repeats for each group; All data are mean ± SD; p-q, unpaired t test; r-s, multiple t tests).
(t-w) qPCR analysis of relative mRNA expression of Sig1R (t), Western Blot analysis and quantification of Sig1R protein expression (u) and qPCR analysis of relative mRNA expression of anti-oxidative (v) and ER stress (w) related genes of Sig1R -knockdown primary SLCs from 2 months old mice. (n = 3 biological repeats for each group; All data are mean ± SD; t-u, unpaired t test; v-w, multiple t tests). Two-sided comparison; Error bars represent SDs. *p < 0.05, **p < 0.01, ***p < 0.001.
Uncropped western blots and source data are provided as a Source Data file.   (a) qPCR analysis of relative mRNA expression of Nestin in Nestinknockdown primary SLCs from 2 months old mice. (n = 3 biological repeats for each group; All data are mean ± SD; One-way ANOVA).
(b-c) Western Blot analysis and quantification of Nestin protein expression of Nestin-knockdown primary SLCs from 2 months old mice. (n = 3 biological repeats for each group; All data are mean ± SD; One-way ANOVA).
(d) Representative immunostaining pictures of colocalization between mitochondria and ER in Nestin-knockdown primary SLCs from 2 months old mice. Mitochondria and ER are marked with Mitotracker and ER tracker, respectively. Scale bar, 10 μm.
(e-f) Quantification of the levels of colocalization in (F) (Manders' (of mitochondria) and Pearson's coefficients are shown for each condition, n = 57, 52 and 45 cells for Control, shNES-1 and shNES-2 group, respectively; All data are mean ± SD; One-way ANOVA).
(g) Flow cytometry of intracellular ROS level stained with DHE of Nestinknockdown primary SLCs from 2 months old mice in vitro. 100 μM antimycin was the positive control added to the Control group.
(h) Quantification of mean fluorescence intensity of DHE in (I). (n = 3 biological repeats for each group; All data are mean ± SD; One-way ANOVA).
(j) Quantification of percentage of SA-β-Gal positive cells in (D). (n = 3 biological repeats for each group; All data are mean ± SD; One-way ANOVA).
(k) qPCR analysis of relative mRNA expression of testosterone production related genes at day 9 during induced differentiation of Nestin-knockdown primary SLCs from 2 months old mice. (n = 3 biological repeats for each group; All data are mean ± SD; Two-way ANOVA).
(m) Quantification of the mean fluorescent intensity in (L) (n = 3 biological repeats for each group; All data are mean ± SD; Multiple t tests).
(n) Quantification of testosterone level in the supernatants of medium during induced differentiation at different time points (day 3, day 6, day 9) of Nestinknockdown primary SLCs from 2 months old mice (n = 3 biological repeats for each group; All data are mean ± SD; Two-way ANOVA). Two-sided comparison; Error bars represent SDs. *p < 0.05, **p < 0.01, ***p < 0.001.
Uncropped western blots and source data are provided as a Source Data file. biological repeats for each group; All data are mean ± SD; Unpaired t test).
(m) Quantification of mitochondrial ATP production in primary SLCs from 2 months old, 24 months old and melatonin-treated 24 months old mice. (n = 3 biological repeats for each group; All data are mean ± SD; Unpaired t test).
(n) Quantification of glucose uptake in primary SLCs from 2 months old, 24 months old and melatonin-treated 24 months old mice. (n = 3 biological repeats for each group; All data are mean ± SD; Unpaired t test).
Uncropped western blots and source data are provided as a Source Data file. (a-f) qPCR analysis of relative mRNA expression of testosterone production related genes in LCs induced by primary SLCs from 2 months old, 24 months old and melatonin-treated 24 months old mice at day 9. (n = 3 biological repeats for each group; All data are mean ± SD; Unpaired t test).
(g) Quantitative analysis of mean fluorescence intensity of LHR staining in Figure. 5g. (n = 3 biological repeats for each group; All data are mean ± SD; Unpaired t test).
(h) Quantitative analysis of testosterone level in the serum from 2 months old, 24 months old and melatonin-treated 24 months old mice in day 9. (n = 3 biological repeats for each group; All data are mean ± SD; Unpaired t test).
(i-j) Western Blot analysis and quantification of Nestin expression in seminiferous tubules from 2 months old, 24 months old and melatonin-treated 24 months old mice after treatment with melatonin for 5 days. (n = 3 biological repeats for each group; All data are mean ± SD; Unpaired t test). Two-sided comparison; Error bars represent SDs. *p < 0.05, **p < 0.01, ***p < 0.001.
Uncropped western blots and source data are provided as a Source Data file.  (n = 5 biological repeats for each group; All data are mean ± SD).
(c) Immunofluorescence analysis was carried out to determine the colocalization of AAV8-GFP (green) and PDGFRα (red). 30 μm for original pictures and 15 μm for enlarged pictures.
(d) Statistical analysis of efficacy of AAV8 transduction in PDGFRα+ SLCs (n = 5 biological repeats for each group; All data are mean ± SD).
(e) qPCR analysis of relative mRNA expression of Nestin. (n = 3 biological repeats for each group; All data are mean ± SD; One-way ANOVA and Sidak's multiple comparisons test) (f) Western Blot analysis of Nestin expression in the testis of wild type, AAV8-sgCon, AAV8-sgNes and AAV8-sgNes+Mel groups.
(i) Quantification of mean fluorescence intensity of DHE in Figure. 7i. (n = 3 biological repeats for each group; All data are mean ± SD; One-way ANOVA and Sidak's multiple comparisons test).